LOCUS       sequence-123943-        3313 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Golden Gate recipient and Gateway entry vector; assembly of 5 gRNAs 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3313)
  AUTHORS   Lowder LG, Paul JW, Baltes NJ, Voytas DF, Zhang Y, Zhang D, Tang X, 
            Zheng X, Hsieh TF, Qi Y
  TITLE     A CRISPR/Cas9 toolbox for multiplexed plant genome editing and 
            transcriptional regulation.
  JOURNAL   Plant Physiol. 2015 Aug 21. pii: pp.00636.2015.
  PUBMED    26297141
REFERENCE   2  (bases 1 to 3313)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Physiol. 2015 Aug 21. pii: pp.00636.2015."
COMMENT     Sequence Label: pYPQ145
FEATURES             Location/Qualifiers
     source          1..3313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     202..218
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        228..246
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(294..314)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(294..312)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(333..349)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(346..368)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    357..373
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(381..387,388..405,406..411))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    426..447
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(512..611)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(629..647)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(652..668)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1099..1890
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1983..2571
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2472..2491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2725..2742
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2901..2928)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(3020..3106)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     3170..3186
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3202..3297
                     /gene="mutant version of attL"
                     /label=attL5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(join(3213..3313,1..337))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGAGLDSH
                     HLGILRYLEGTPWGNFCIQSWHYKKALLINLLQRTGHYQSK"
ORIGIN
        1 ctagataacg caggatcccc aagtggtggc tatcgagacc ggcgccgcta cagggcgcgt
       61 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
      121 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      181 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
      241 tatagggcga attgggtacc gggccccccc tcgaggtcct ccagcttttg ttccctttag
      301 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      361 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      421 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccaccgg
      481 tggtctcttg caactagtaa gggcgaattc gacccagctt tcttgtacaa agttggcatt
      541 ataaaaaata attgctcatc aatttgttgc aacgaacagg tcactatcag tcaaaataaa
      601 atcattattt gccatccagc tgatatcccc tatagtgagt cgtattacat ggtcatagct
      661 gtttcctggc agctctggcc cgtgtctcaa aatctctgat gttacattgc acaagataaa
      721 aatatatcat catgcctcct ctggaccagc caggacagaa atgcctcgac ttcgctgctg
      781 cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac ccagtggaca
      841 taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg caactggtcc
      901 agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat
      961 gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc
     1021 gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct
     1081 aaaacaaagt taaacattat gagggaagcg gtgatcgccg aagtatcgac tcaactatca
     1141 gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac
     1201 ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct ggttacggtg
     1261 accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg
     1321 gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac
     1381 gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc
     1441 aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg
     1501 ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt
     1561 gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac
     1621 tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg
     1681 tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag
     1741 cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa
     1801 gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca ctacgtgaaa
     1861 ggcgagatca ccaaggtagt cggcaaataa ccctcgagcc acccatgacc aaaatccctt
     1921 aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     1981 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2041 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2101 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     2161 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2221 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2281 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2341 acctacaccg aactgagata cctacagcgt gagcattgag aaagcgccac gcttcccgaa
     2401 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2461 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2521 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2581 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     2641 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     2701 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     2761 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     2821 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atacgcgtac cgctcgccag
     2881 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
     2941 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
     3001 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
     3061 taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc
     3121 cctactctcg cgttaacgct tgcatggatg ttttcccagt cacgacgttg taaaacgacg
     3181 gccagtctta agctcgggcc caaataatga ttttattttg actgatagtg acctgttcgt
     3241 tgcaacaaat tgatgagcaa tgctttttta taatgccaac tttgtataca aaagttgccc
     3301 catggcgttc cct
//
